A 2.5-kilobase fragment of a sex-specific satellite DNA from the Colubrid snake species Elaphe radiata has been cloned, and its sequence has been determined. It contains 26 and 12 copies, respectively, of two base quadruplets, G-A-T-A and G-A-C-A, as its sole highly repetitious elements. Southern hybridization experiments with genomic DNA of the chicken, the mouse, and man indicated male sex-specific conservation of at least parts of this cloned DNA. In situ hybridization experiments with metaphase chromosomes of the mouse showed that elements that can cross-hybridize with parts of the cloned snake DNA are concentrated in the pericentric region of the Y chromosome. In blot hybridization experiments with liver poly(A)+ polysomal RNAs of male and female mice, a probe consisting of the first 1,224 bases of the cloned snake DNA singled out a male-specific RNA of 1,250-1,400 bases. Inasmuch as the proximal end of this probe contained an open reading frame (44 consecutive amino acid-specifying codons), the male-specific putative mRNA so detected may specify H-Y antigen. By contrast, a probe consisting of bases 1,480-1,906, containing the simple repeats of the quadruplets, singled out a shorter (approximately 1,000-base) RNA from males and females alike. Although this RNA is poly(A)+, we have yet to establish its attachment to ribosomes.